β-ketoacyl-CoA reductase KCR gene and its application in improving plant salt tolerance

Abstract

The present invention relates to the technical field of plant genetic engineering. The present invention provides a β-ketoacyl-CoA reductase KCR gene, and the nucleotide sequence of the KCR gene is shown in SEQ ID NO.1. The present invention also provides a recombinant vector. The present invention also provides a recombinant bacterium. The present invention also provides the application of the KCR gene, recombinant vector or recombinant bacterium as described above in improving the salt tolerance of plants. The present invention also provides the application of the KCR gene, recombinant vector or recombinant bacterium as described above in the genetic improvement of plant salt tolerance. The present invention also provides a method for obtaining salt-tolerant plant strains. After overexpressing the KCR gene in grape callus, the salt tolerance of the obtained transgenic grape callus is significantly increased. After overexpressing the gene in Arabidopsis thaliana, the epidermal wax accumulation of the obtained transgenic Arabidopsis thaliana is increased while the salt tolerance is significantly increased, which can provide new gene resources for the salt tolerance improvement and germplasm resource selection of grape cultivars.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to β-ketoacyl-CoA reductase KCR Genes and their application in improving plant salt tolerance. Background Art

[0002] Grape( Vitis vinifera L. Grapes are one of the world's top ten fruits, boasting strong adaptability and high economic returns, making them a widely cultivated fruit variety in my country. Soil salinization severely impacts plant physiological activities such as photosynthesis, oxidative balance, and transpiration, leading to reduced grape yield and quality. Therefore, improving grapes' adaptability to salinized soils and achieving salt-tolerant cultivation are critical challenges in grape production.

[0003] Under salt stress, multiple physiological and biochemical processes in plants change, ultimately affecting their growth and development. Under salt stress, plants respond by maintaining ion homeostasis, accumulating soluble substances, and enhancing antioxidant defense systems. Salt-alkali stress affects ion transport, preventing plants from providing energy and thus inhibiting photosynthesis. Plant osmotic regulators, such as soluble sugars, soluble proteins, and proline, regulate intracellular osmotic pressure, improve water retention, and maintain normal plant physiological metabolism. Both biotic and abiotic stresses can trigger the production of reactive oxygen species (ROS). Under salt stress, ROS levels increase dramatically, and excessive ROS can cause significant damage to proteins, lipids, and nucleic acids, leading to cell damage and even death. Furthermore, salt-alkali stress can induce the expression of relevant resistance genes, which are primarily involved in osmotic regulation, ion homeostasis, oxidative activity, and hormone signal transduction.

[0004] Plant cuticle wax is a waxy substance covering the epidermis of terrestrial plants. It is an important barrier between plants and the external environment. It plays an important role in maintaining water balance, resisting pests and diseases, preventing mechanical damage, reducing ultraviolet radiation and maintaining fruit quality. Cuticle wax is mainly composed of membrane very long-chain fatty acids (VLCFAs), alcohols, aldehydes, esters and alkane organic matter. Its synthesis process involves the coordinated participation of multiple enzymes, mainly including long-chain acyl-CoA synthetase, β-ketoacyl-CoA synthase, β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydratase, fatty acyl-CoA reductase and wax ester synthase. The corresponding coding gene is LACS1, KCS1, KCR1, CER1, CER2, CER4 and WSD1 wait.

[0005] In plants, the biosynthesis of very-long-chain fatty acids (VLCFAs) is divided into two stages. The first stage is de novo synthesis of fatty acids in plastids, which is completed in four steps: condensation, reduction, dehydration, and reduction under the catalysis of fatty acid synthases (FAS), forming free fatty acids. The second stage is carbon chain elongation. The carbon chain elongation enzyme is a multi-enzyme complex composed of four different enzymes: β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD), and trans-2,3-enoyl-CoA reductase (ECR). These four enzymes are involved in the process of fatty acid carbon chain elongation in sequence, and ultimately generate very-long-chain fatty acids. VLCFAs play an important role in plant growth and development and in response to various stresses. In jute, CcNAC1 interacts with KCS, ultimately promoting early flowering and enhancing drought tolerance. AKR2A can affect the biosynthesis of VLCFAs in Arabidopsis by interacting with KCS1, thus participating in the regulation of cold tolerance. Cotton GbKCS14 Synthesize VLCFAs in response to low temperature at the initial stage of lint formation in Gossypium barbadense L. However, there is currently no research on the relationship between grape KCR and plant salt tolerance. Summary of the Invention

[0006] The object of the present invention is to provide a β-ketoacyl-CoA reductase KCR (NCBI Gene ID accession number: PP588439) gene and its application in improving plant salt tolerance. The present invention first clones the grape KCR gene, analyzes its expression characteristics, then observes its distribution in cells through subcellular localization, constructs a plant expression vector, transforms grape callus and Arabidopsis, identifies its positive seedlings, and analyzes its salt tolerance through salt stress treatment. Experiments show that overexpression of KCR the gene makes transgenic grape callus have higher salt tolerance.

[0007] To solve the above problems, the present invention provides the following technical solutions:

[0008] In the first aspect, the present invention provides a β-ketoacyl-CoA reductase KCR gene (which may be abbreviated as " KCR gene" or " KCR”), and the nucleotide sequence of the β-ketoacyl-CoA reductase KCR gene is as shown in SEQ ID NO.1. This gene contains an open reading frame of 963 bp and 320 amino acids. The KCR gene is salt-induced and plays a role in plant salt stress tolerance. The molecular formula composition of the KCR gene is C 1622 H 2584 N 426 O 436 S 13 , with a predicted molecular weight of 3.54 kDa, an isoelectric point of 8.59, and an instability index of 26.37.

[0009] The β-ketoacyl-CoA reductase KCR gene as described above, and the protein sequence encoded by the β-ketoacyl-CoA reductase KCR gene is as shown in SEQ ID NO.2.

[0010] The KCR amplification primers, the forward primer is as shown in SEQ ID NO.3, and the reverse primer is as shown in SEQ ID NO.4.

[0011] In a second aspect, the present invention provides a recombinant vector carrying the β-ketoacyl-CoA reductase KCR gene as described above.

[0012] In a third aspect, the present invention provides a recombinant bacterium comprising the recombinant vector described above.

[0013] In a fourth aspect, the present invention provides an application of the β-ketoacyl-CoA reductase KCR gene as described above, the recombinant vector as described above, or the recombinant bacterium as described above in improving the salt tolerance of plants.

[0014] In the present invention, salt tolerance refers to the tolerance of plant materials to 100 - 250 mM NaCl salt treatment.

[0015] The application as described above is at least one of the following (1) - (4):

[0016] (1) Transient overexpression of the KCR gene in plants significantly increases the accumulation of phenols, wax esters, and alkanes in leaves;

[0017] (2) Transient silencing of the KCR gene in plants significantly reduces the accumulation of phenols, wax esters, and alkanes in leaves;

[0018] (3) Transient overexpression of the KCRThe gene was overexpressed in grape callus. Under salt stress, the overexpressed grape callus had increased activities of SOD, POD, and CAT compared to the wild-type grape callus; the content of MDA, the accumulation of H2O2, and the superoxide anion were decreased;

[0019] (4) The KCR gene was overexpressed in grape callus. Under salt stress, the expression level of the salt-responsive gene CAX, HATP, HKT2, HPP, SOS1, SORK was significantly increased.

[0020] The application as described above, wherein the plant material used for transient transformation is the leaves of 'Zuoyouhong' grape, and the grape callus is induced from 'Thompson Seedless' grape. In the present invention, transient transformation refers to

[0021] Fifthly, the present invention provides an application of the β-ketoacyl-CoA reductase KCR gene as described above, the recombinant vector as described above, or the recombinant bacterium as described above in the genetic improvement of plant salt tolerance. Preferably, the plant is the experimental material used for transient transformation is 'Zuoyouhong' grape, the experimental material used for studying the salt tolerance of grape callus is the callus of 'Thompson Seedless' grape, and Arabidopsis thaliana.

[0022] The application as described above, the application is at least one of the following (a) to (b):

[0023] (a) Overexpress the KCR gene in callus, and induce the callus to form seedlings;

[0024] (b) Overexpress the KCR gene in the plant, and propagate and screen the offspring.

[0025] Sixthly, the present invention provides a method for improving plant salt tolerance. The nucleotide sequence SEQ ID NO.1 of the β-ketoacyl-CoA reductase KCR gene as described above is constructed into an expression vector to form a recombinant expression vector, and then the recombinant expression vector is transformed into a strain to obtain a recombinant strain carrying the KCR gene. Then, the recombinant strain is used to infect the plant, so that the plant carries the KCR gene, and finally, through the KCR gene expression, the accumulation of epidermal wax and the biosynthesis of VLCFAs are regulated, thereby regulating the salt tolerance of the plant. Optionally, the plant part of the recombinant strain infecting the plant is grape leaves, grape callus, or Arabidopsis thaliana plants. Optionally, the plant is 'Zuoyouhong' grape.

[0026] Compared with the existing technology, the effects and advantages of the present invention are:

[0027] 1. In the present invention, theKCR After the gene was overexpressed in grape callus, the salt tolerance of the obtained transgenic grape callus increased significantly. After the gene was overexpressed in Arabidopsis thaliana, the epidermal wax accumulation of the obtained transgenic Arabidopsis thaliana increased simultaneously, and the salt tolerance increased significantly, which could provide new gene resources for the improvement of salt tolerance and the selection and breeding of germplasm resources of grape cultivars.

[0028] 2. The β-ketoacyl-CoA reductase KCR of the present invention participates in the response of grapes to salt stress by regulating epidermal wax synthesis, excavates the very long-chain fatty acids (VLCFAs) and key genes for epidermal wax synthesis in grape salt stress response, analyzes its biological functions and mechanisms of action, uses relevant gene resources to improve the salt tolerance of grape cultivars and select and breed germplasm resources, and develops regulatory technologies to enhance the salt resistance of fruit trees, which is of great significance for realizing the improvement of quality and efficiency of salt-tolerant cultivation of grapes and other fruit trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the technical flow chart of the present invention;

[0030] Figure 2 of the present invention KCR Gene expression characteristic analysis diagram: A is the expression characteristics in different tissue organs (roots, buds, tendrils, stems, leaves, fruits) of grapes; B is the expression characteristics at different treatment times;

[0031] Figure 3 is the schematic diagram of subcellular localization of KCR of the present invention;

[0032] Figure 4 of the present invention KCR Schematic diagram of the morphology and content of epidermal wax on grape leaves under salt stress by transient overexpression and transient silencing (WT is the wild-type grape leaf; OEKCR is the grape leaf with transient overexpression of KCR; pTRV2-KCR is the grape leaf with transient silencing of KCR); wherein: A is the morphology of epidermal wax on wild-type, transient overexpression KCR and transient silencing KCR grape leaves under non-salt stress and salt stress; B is the content of each component of VLCFAs in wild-type, transient overexpression KCR and transient silencing KCR grape leaves under non-salt stress and salt stress; C is the content of epidermal wax on wild-type, transient overexpression KCR and transient silencing KCR grape leaves under non-salt stress and salt stress;

[0033] Figure 5 of the present invention KCR Schematic diagram of the phenotype and identification of salt-treated overexpressed grape callus; wherein: A is the phenotype of overexpressed grape callus and wild-type grape callus before and after salt treatment; B is KCR ​KCR Identification of overexpressed grape callus; C is a schematic diagram of the content of each component of VLCFAs;

[0034] Figure 6 For the present invention KCR Schematic diagram of the determination of physiological indexes of overexpressed grape callus under salt treatment; wherein: A is the malondialdehyde content of grape callus before and after low-temperature treatment; B is the superoxide anion content of grape callus before and after salt treatment; C is the hydrogen peroxide content of grape callus before and after salt treatment; D is the SOD activity of grape callus before and after salt treatment; E is the POD activity of grape callus before and after salt treatment; F is the CAT activity of grape callus before and after salt treatment;

[0035] Figure 7 A-F are the expression levels of salt-responsive genes ( CAX, HATP, HKT2, HPP, SOS1, SORK ) of grape callus before and after salt treatment;

[0036] Figure 8 For the present invention KCR Heterologous overexpressed Arabidopsis plants; wherein: A is the screening of overexpressed Arabidopsis using kanamycin; B is KCR Detection of the expression level of heterologous overexpressed Arabidopsis; C is KCR Identification of heterologous overexpressed Arabidopsis lines;

[0037] Figure 9 For the present invention KCR Schematic diagram of the phenotype and survival rate of heterologous overexpressed Arabidopsis plants under salt treatment; wherein: A is the phenotype and germination rate of seeds of heterologous overexpressed Arabidopsis before and after salt treatment; B is the phenotype and main root length of seedlings of heterologous overexpressed Arabidopsis and wild-type Arabidopsis seedlings before and after salt treatment; C is the phenotype of adult plants of heterologous overexpressed Arabidopsis and wild-type Arabidopsis adult plants before and after salt treatment;

[0038] Figure 10 For the present invention KCR Epidermal wax morphological structure and content of adult plants of heterologous overexpressed Arabidopsis; wherein: A is the epidermal wax morphological structure of Arabidopsis before and after salt treatment; B is the content of each component of VLCFAs of Arabidopsis before and after salt treatment; C is the epidermal wax content of Arabidopsis before and after salt treatment;

[0039] Figure 11 For the present invention KCR Schematic diagram of the determination of physiological indexes of adult plants of heterologous overexpressed Arabidopsis under salt treatment; wherein: A is the relative water content of Arabidopsis before and after salt treatment; B is the leaf water loss rate of Arabidopsis before and after salt treatment;

[0040] Figure 12A is the relative permeability of the cell membrane of Arabidopsis thaliana before and after salt treatment; B is the malondialdehyde content of Arabidopsis thaliana before and after salt treatment; C is the superoxide anion content of Arabidopsis thaliana before and after salt treatment; D is the SOD activity of Arabidopsis thaliana before and after salt treatment; E is the POD activity of Arabidopsis thaliana before and after salt treatment; F is the CAT activity of Arabidopsis thaliana before and after salt treatment. Detailed implementation manners

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the content in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific implementation manners and are not used to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0043] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0044] Figure 1 This is the technical flow chart of the present invention. For the grape KCR gene, cloning is carried out, its expression characteristics are analyzed, and then its distribution in cells is observed through subcellular localization. A plant expression vector is constructed, grape callus and Arabidopsis thaliana are transformed, and its positive seedlings are identified. Through salt stress treatment, its salt tolerance is analyzed.

[0045] Example 1 KCR cloning

[0046] Grape KCR For the cloning of the full-length cDNA of the gene, the cDNA of Vitis vinifera cv. Zuoyouhong was used as a template, and high-fidelity enzyme was used for amplification. The sequences of the amplification primers were SEQ ID NO. 3 (forward primer) and SEQ ID NO. 4 (reverse primer) as follows:

[0047] Forward primer: 5’-ATGGAACTTTGTTTCATGGAAAAGCTCC -3’,

[0048] Reverse primer: 5’-CTCCTGCTTCCTAGACCCTTTGAGTT -3’,

[0049] The amplified product was purified and recovered using a gel extraction kit. Using DNA cloning technology, the purified product was ligated into the pTOPO-Blunt vector (purchased from Beijing Polymer Beauty Biotechnology Co., Ltd.), and then the ligation product was transformed into DH5α competent cells (purchased from Sangon Biotech Co., Ltd.). The cells were plated, cultured in a shaker, and then subjected to positive identification. After obtaining positive clones, they were sent to a biological company for sequencing. According to the sequencing results, the KCR full-length gene sequence was obtained. The size of the KCR sequence in the sequencing result was 963 bp, encoding 320 amino acids. The molecular formula of KCR was C 1622 H 2584 N 426 O 436 S 13 , with a predicted molecular weight of 3.54 kDa, an isoelectric point of 8.59, and an instability index of 26.37.

[0050] The sequenced CDS sequence was SEQ ID NO.1:

[0051] ATGGAACTTTGTTTCATGGAAAAGCTCCAAACTCAGCCACTCTGGGTTATTGTGCTCTTTGCTGTGGGATGTTTATCGATTTTGAAATCTTTCCTAGCTATCCTCAATGGGGTCTACGTTTGTTTTCTCAGACCGGGCAAGAATCTCAAGAAATACGGGTCGTGGGCACTCGTGACTGCTCCCACGGACGGTATCGGAAAGGGTTTTGCCTTTGAATTGGCTCGAAAAGGGCTTAATCTGGTATTGGTGGGTCGGAATCCGGATAAGCTGAAGGATGTGTCCGATGCGATTCAATCCAAATATGGGAAGACCCAGATCAAGGCTGTTGTGGTTGATTTTGCTGGTGATATTTCCGAGGGTGTTCTGAAGATTCGAGAAGCTATTGAAGGACTGGATGTTGGGGTTTTGATCAATAATGTGGGGGTTTCATATCCATATGCCAGGTTTTTTCATGAAGTGGATGATGAGCTGTTGAAGAATTTGATTAAAGTGAATGTCGAGGGCACCACGAAGGTCACACAGGCTGTTCTTCCAGGGATGCTCAAGAGGAAGAAGGGTGCAATTGTCAATATTGGTTCAGGTGCTGCTATTGTGATTCCTTCAGATCCTCTTTATGCTGTTTATGCGGCCACAAAAGCGTATATTGATCAATTCTCAAGGTGCCTCTATGTTGAATATAAGAATAGCGGGATTGATGTGCAGTGTCAGGTTCCACTGTATGTGGCCACTAAGATGGCATCAATCAGGAGATCTTCCTTCTTAGTTCCCTCATCAGATGGCTATGCCCGTGCAGCCATTCGCTGGATAGGGTATGAACCACGCTGCACTCCTTATTGGCCCCATGTCCTTATCTGGGGTCTGGCATGCCAATTACCAGAGTGTGCTATTGATGCATGGCGCCTGGGATTCAACCTTAAGATTAGAAAGAGGGGACAACTCAAAGGGTCTAGGAAGCAGGAGTAG。

[0052] The encoded amino acid sequence is: SEQ ID NO.2:

[0053] MELCFMEKLQTQPLWVIVLFAVGCLSVLKSFLAILNGVYVCFLRPGKNLKKYGSWALVTAPTDGIGKGFAFELARKGLNLVLVGRNPDKLKDVSDAIQSKYGKTQIKAVVVDFAGDISEGVLKIREAILGLDVGVLINNVGVSYPYARFFHEVDDELLKNLIKVNVEGTTKVTQAVLPGMLKRKKGAIVNIGSGAAIVIPSDPLYAVYAATKAYIDQFSRCLYVEYKNSGIDVQCQVPLYVATKMASIRRSSFLVPSSDGYARAAMRWIGYEPRCTPYWPHVLIWGLACQLPECAIDVWRLGFNLKIRKRGQLKGSRKQE.

[0054] Example 2 Analysis of expression characteristics under salt treatment KCR

[0055] 1. Experimental method:

[0056] Take one-month-old grape Zuoyouhong and Shuangyou resistant tissue culture seedlings, and Chardonnay and Cabernet Sauvignon sensitive tissue culture seedlings cultivated in a cultivation room with a light intensity of 200 mmol / (m2·s), a light / dark cycle of 16 / 8 h, and a temperature of 25±1°C. Treat them with 150 mM NaCl by soaking their roots. The sampling time points are 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h. After taking the leaves at each time point, quickly put them into liquid nitrogen for freezing, and then store them in a refrigerator at 80°C for later use in gene expression pattern analysis.

[0057] Select one-year-old branches of "Zuoyouhong" for different tissue organs (roots, stems, buds, tendrils, leaves, and fruits) of grapes. Take 0.1 g of each and put them into liquid nitrogen for freezing, and then store them in a refrigerator at 80°C for later use in tissue expression characteristic analysis.

[0058] Extract total RNA from grapes by the CTAB method, and synthesize the first strand of cDNA as a template using an M-MLV reverse transcription kit (purchased from Shanghai Shangbao Biotechnology Co., Ltd.). Analyze the salt treatment and tissue expression characteristics of the KCR gene by real-time fluorescence quantitative PCR (qRT-PCR). The Real timePCR program is: 95°C for 60 s, 95°C for 10 s, 58°C for 20 s, 72°C for 15 s, for 40 cycles. Each sample is repeated 3 times, and the reaction is carried out using a TaKaRa PCR fluorescence quantitative analyzer.

[0059] Using Actin in grapes as an internal reference gene (forward primer: 5’ ATAGAAGCAGCAAGGGA 3’; reverse primer: 5’ TGAGGCTCTTACTAATG 3’), the 2 -△△CT algorithm was used to calculate gene expression.

[0060] KCR real-time quantitative primers (forward primer: 5’ TGTGCTCTTTGCTGTGGGAT 3’; reverse primer: 5’GCCCACGACCCGTATTTCTT 3’).

[0061] 2. Experimental results and analysis:

[0062] From Figure 2 it can be seen that KCR Analysis of tissue expression characteristics found that Figure 2 A shows that KCR the expression levels are significant in leaves and fruits, Figure 2 B shows that the expression level in resistant varieties is significantly higher than that in sensitive varieties.

[0063] The above results indicate that KCR the gene is induced by salt stress and may play a role in grape salt stress tolerance.

[0064] Example 3 Subcellular localization analysis of KCR

[0065] 1. Experimental method:

[0066] Using the plasmid of pTOPO-Blunt-KCR as a template to amplify the CDS fragment of KCR, and fusing it into the vector pSuper1300GFP (purchased from Shanghai Beinuo Biotechnology Co., Ltd.), then transforming the recombinant plasmid of pSuper1300 KCR GFP into Agrobacterium tumefaciens GV3101 (purchased from Sangon Biotech Co., Ltd.) and performing colony PCR identification, and selecting positive bacteria for subsequent experiments. Inject the constructed pSuper1300 KCR GFP and the positive control pSuper1300 GFP into tobacco leaves respectively, and observe the subcellular localization of KCR with a laser confocal microscope after 3 days of expression.

[0067] 2. Experimental results and analysis:

[0068] From Figure 3 it can be seen that KCR is distributed on the endoplasmic reticulum.

[0069] Example 4 Transient overexpression KCR Cuticular wax morphology and content of grape leaf epidermis

[0070] Cut the leaves of grape tissue culture seedlings, and rinse them twice with sterile water for transient transformation and infection. The operation is as follows:

[0071] (1) Streak p19 (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.), pSuper1300 GFP, and the recombinant vector of the target gene Agrobacterium tumefaciens GV3101 on LB (containing 50 mg / L Rif, 50 mg / L Kan) solid medium respectively, and invert and culture at 28 °C for 2 - 3 days to obtain monoclonal colonies;

[0072] (2) Pick one monoclonal colony into 10 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan respectively, and culture at 28 °C, 220 r / min, with gentle shaking for 12 - 24 h to fully activate the bacteria;

[0073] (3) Inoculate the activated Agrobacterium tumefaciens liquid into the same LB liquid medium at a volume ratio of 1:30, culture at 28 °C, 220 r / min, expand the culture for 10 - 12 h, then centrifuge at 4000 r / min to collect the bacteria, add MES buffer (10 mmol / L MES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6 - 5.7) to suspend the bacteria, and adjust OD600 to 1.0;

[0074] (4) Mix p19 and pSuper1300 GFP, p19 and the target gene bacterial liquid at a volume ratio of 1:1 respectively, mix well and culture in the dark at 28 °C for 2 - 3 h to prepare the infection solution;

[0075] (5) Gently prick some small holes on the grape leaves with a syringe needle, soak them completely in the prepared Agrobacterium tumefaciens infection solution, perform vacuum pumping for 10 min, quickly release the air to make Agrobacterium tumefaciens invade the grape leaves, and repeat 3 times. Then let it stand for 15 min, take out the infected leaves and wipe the bacterial liquid, lay them flat in a large dish with filter paper moistened with sterile water. Culture at room temperature, place in the dark for 1 day and then culture under normal light for 2 - 3 days, and then carry out subsequent experiments.

[0076] Example 5 Transient silencing of the morphology and content of epidermal wax of KCR grape leaves

[0077] 1. Vector construction

[0078] Use the plasmid of pTOPO - Blunt - KCR as a template to amplify the CDS fragment of KCR, and ligate it to the pTRV2 vector (purchased from Changsha Abiwei Biotechnology Co., Ltd.). After the constructed vector is correctly sequenced, it is transferred into GV3101 competent cells.

[0079] 2. VIGS infection

[0080] Cut the leaves of grape tissue - cultured seedlings, and rinse them twice with sterile water for transient transformation and infection. The operation is as follows:

[0081] (1) Streak pTRV1 (purchased from Coolaber Technology Co., Ltd., Beijing), pTRV2 (purchased from Coolaber Technology Co., Ltd., Beijing), and the Agrobacterium tumefaciens GV3101 recombinant vector carrying the target gene on LB (containing 50 mg / L Rif and 50 mg / L Kan) solid medium respectively, and incubate them in an inverted position at 28 °C for 2 - 3 d to obtain monoclonal colonies;

[0082] (2) Pick one monoclonal colony into 10 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan, and incubate it at 28 °C with shaking at 220 r / min for 12 - 24 h to fully activate the bacteria;

[0083] (3) Inoculate the activated Agrobacterium tumefaciens liquid into the same LB liquid medium at a volume ratio of 1:30, incubate it at 28 °C with shaking at 220 r / min for 10 - 12 h, then centrifuge at 4000 r / min to collect the bacteria. Add MES buffer (10 mmol / L MES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6 - 5.7) to suspend the bacteria, and adjust the OD600 to 1.0;

[0084] (4) Mix pTRV1 and pTRV2, and pTRV1 and the target gene bacterial liquid at a volume ratio of 1:1 respectively. After mixing, incubate them in the dark at 28 °C for 2 - 3 h to prepare the infection solution;

[0085] (5) Gently prick some small holes on the grape leaves with a syringe needle, completely immerse them in the prepared Agrobacterium tumefaciens infection solution, perform vacuum pumping for 10 min, quickly release the air to allow the Agrobacterium tumefaciens to invade the grape leaves, and repeat 3 times. Then let it stand for 15 min, take out the infected leaves, wipe the bacterial liquid, and spread them flat in a large dish with filter paper moistened with sterile water. Incubate at room temperature, place them in the dark for 1 d, then culture them under normal light for 2 - 3 d, and then carry out subsequent experiments.

[0086] Example 6 Transient transformation of the morphology and content of epicuticular wax on grape leaves of KCR

[0087] Obtain transient overexpression grape leaves of pSuper1300 - KCR - GFP and transiently silenced grape leaves of pTRV2 - KCR by Agrobacterium tumefaciens infection, and perform 150 mM NaCl salt treatment on them. Use scanning electron microscopy and gas chromatography to analyze the differences in VLCFAs and epicuticular wax content on leaves. The results are as Figure 4 shown.

[0088] From Figure 4It can be seen that under normal growth conditions, there were no significant differences in the morphology and content of epidermal wax between transiently transformed grape leaves and the empty vector. However, after salt stress treatment, compared with the wild type, the content of VLCFAs in transiently overexpressing KCR grape leaves increased significantly, and the components of VLCFAs, especially the contents of C22 and C24, increased significantly. Transient silencing KCR of grape leaves significantly reduced the content of VLCFAs, and the components of VLCFAs, especially the contents of C22 and C24, decreased significantly ( Figure 4 B). After salt stress treatment, compared with the wild type, transient overexpression KCR of grape leaves significantly increased the epidermal wax crystals, and the content of epidermal wax increased significantly ( Figure 4 A, the arrow indicates the epidermal wax crystals), among which phenols, wax esters and alkanes increased significantly, while transient silencing KCR of grape leaves significantly reduced the epidermal wax crystals, and the content of epidermal wax decreased, especially the relative reduction of phenols, wax esters and alkanes ( Figure 4 C).

[0089] The above results indicate that KCR regulates the synthesis of VLCFAs and epidermal wax and participates in the process of plant resistance to salt stress.

[0090] Example 7 Genetic transformation of grape callus and identification of overexpressing callus

[0091] 1. Genetic transformation of grape callus

[0092] (1) Preparation of plant materials

[0093] Using the flower buds of grape variety 'Thompson Seedless' as experimental materials, cut them into small pieces and put them into a sterilized empty bottle, disinfect with 75% alcohol for 30 s, soak and disinfect with 15 - 18% NaClO for 15 min, and wash with sterile water 3 - 5 times. After disinfection, place them on the induction medium and culture them in the dark in a 25°C light incubator. The induced grape embryogenic callus was inoculated on the subculture medium and cultured in the dark in a 25°C light incubator, and was subsequently used for obtaining transgenic callus, observing the low-temperature phenotype and detecting stress resistance physiological indexes. During this period, the Agrobacterium liquid was prepared.

[0094] (2) Preparation of Agrobacterium infection solution

[0095] In a laminar flow hood, use a sterilized inoculation loop to pick up Agrobacterium tumefaciens pSuper1300-KCR-GFP stored at -80°C, streak it on a medium containing 50 mg / L Kan and Rif antibiotics, and place the medium in a 28°C incubator for dark incubation for 2 days. Take a sterilized 100 mL conical flask, pour 50 mL of liquid medium containing Kan and Rif antibiotics into it, scrape the grown Agrobacterium tumefaciens and dissolve it in the liquid medium, and shake it at 28°C and 200 r / min for 12 h. Collect the bacterial cells, and add liquid medium containing 20 mg / L AS (Acetosyringone) to adjust the concentration to an OD600 value of 0.6 - 0.8.

[0096] (3)Infection and co-culture

[0097] Place the well-grown grape embryogenic callus in a petri dish containing filter paper, use a pipette to drop the prepared Agrobacterium tumefaciens solution onto the grape callus, and after standing for 5 min, take the filter paper to absorb the residual Agrobacterium tumefaciens solution to complete the infection. Place the grape callus after removing the residual bacterial solution evenly on the co-culture medium containing AS. Then, carry out dark incubation at 25°C for 3 days under shading.

[0098] (4)Screening culture and regeneration

[0099] Take out the grape embryogenic callus after 3 days of dark incubation and transfer it to a screening medium containing 50 mg / L Hyp.

[0100] The formula of the medium used in the experiment is shown in Table 1 below. The prepared medium should be autoclaved at 115°C for 20 min, and after adding filter-sterilized antibiotics in a laminar flow hood, it should be dispensed into petri dishes or conical flasks that have also been autoclaved at 115°C for 20 min, sealed and reserved. The results are as Figure 5 shown.

[0101] Table 1 Medium formula

[0102]

[0103] 2. Identification of positive seedlings

[0104] From Figure 5 It can be seen that by real-time fluorescence quantitative analysis of the relative expression level of KCR in the overexpressed callus, it was found that compared with the wild type, the relative expression level of KCR in the overexpressed callus was significantly increased ( Figure 5 B). The obtained positive transgenic callus was used for subsequent salt tolerance analysis.

[0105] Example 8 Salt tolerance analysis of overexpressed grape callus

[0106] Before salt treatment, there were no obvious phenotypic differences between wild-type and overexpressing grape calli. However, after treatment with 0, 50, 100, and 150 mM NaCl for 7 days, the phenotypes were observed, and the VLCFAs of wild-type and KCR overexpressing calli were detected by gas chromatography.

[0107] From Figure 5 it can be seen that after treatment with 100 mM NaCl, most of the wild-type grape calli turned brown and black, and their growth was significantly inhibited. In contrast, the color of the overexpressing grape calli remained golden yellow, and the degree of damage was smaller ( Figure 5 A). Under salt stress, the contents of each component of VLCFAs increased, and KCR the increase in overexpressing calli was significantly higher than that in wild-type, especially the significant increase in C24 and C26, suggesting KCR that it promoted the accumulation of VLCFAs under salt stress ( Figure 5 C).

[0108] The above results suggest KCR that it promoted the accumulation of VLCFAs under salt stress and participated in the grape salt stress response.

[0109] Under normal conditions, there were no significant differences in the physiological indexes between wild-type and KCR overexpressing calli. From Figure 6 it can be seen that after treatment with 100 mM NaCl, compared with wild-type calli, KCR the contents of SOD, POD, and CAT in overexpressing grape calli increased significantly ( Figure 6 D-F), while the contents of MDA, superoxide anion, and hydrogen peroxide decreased ( Figure 6 A-C).

[0110] This indicates that overexpressing KCR can promote the accumulation of osmoregulatory substances in transgenic materials, reduce the content of ROS, decrease the lipid peroxidation of cell membranes, and improve the salt stress resistance of grapes.

[0111] From Figure 7 it can be seen that in addition, before and after salt treatment, there were no obvious differences in the relative expression levels of salt-responsive genes between wild-type grape calli and overexpressing grape calli. However, after salt treatment, compared with wild-type grape calli, the expression of salt-responsive genes ( CAX, HATP, HKT2, HPP, SOS1, SORK ) in overexpressing grape calli was significantly up-regulated ( Figure 7 in A-F, the first from the left in each group is the wild-type grape calli without salt treatment, the second is the overexpressing KCR grape calli without salt treatment, the third is the wild-type grape calli with salt treatment, and the fourth is the overexpressing KCR grape calli with salt treatment).

[0112] In summary, through the observation of salt tolerance phenotypes and the determination of physiological indexes, it is shown that overexpression of KCR gene endows the transgenic grape callus with higher salt tolerance.

[0113] Example 9 Identification of positive seedlings of overexpressed Arabidopsis thaliana

[0114] Using the "floral dip method", KCR was introduced into Arabidopsis thaliana through Agrobacterium tumefaciens, and then the obtained first-generation seeds were screened using a medium containing kanamycin ( Figure 8 A), real-time fluorescence quantitative analysis of the relative expression level of KCR in overexpressed Arabidopsis thaliana plants was carried out. The results showed that the relative expression level of KCR in overexpressed Arabidopsis thaliana plants was significantly increased compared with that of the wild type ( Figure 8 B), and the heterologous overexpressed Arabidopsis thaliana lines were identified by semi-quantitative analysis ( KCR C). The obtained positive transgenic Arabidopsis thaliana plants were used for subsequent salt tolerance analysis. Homozygous lines of Arabidopsis thaliana with different expression levels were selected for subsequent experiments. They were named Figure 8 and KCR respectively. OEKCR-3, OEKCR-7 and OEKCR-9 .

[0115] Example 10 Salt tolerance analysis of overexpressed Arabidopsis thaliana

[0116] To further verify the biological function of KCR in salt stress response, KCR was heterologously overexpressed in Arabidopsis thaliana. Seeds of wild type and KCR heterologously overexpressed Arabidopsis thaliana, and wild type and KCR heterologously overexpressed Arabidopsis thaliana seedlings that germinated and grew for 14 d after sowing were treated with 100 mM NaCl. The phenotypes were observed and the related physiological indexes were detected; wild type and KCR heterologously overexpressed Arabidopsis thaliana adult plants at about 4 weeks old and without bolting were treated with 250 mM NaCl for 7 d. The phenotypes were observed and the related physiological indexes were detected, and the differences in VLCFAs and leaf epidermal wax content were analyzed by scanning electron microscopy and gas chromatography. The results are as Figure 9 shown.

[0117] As can be seen from Figure 9 , it was found that under normal conditions, there was no obvious difference in the morphology between the wild type and heterologously overexpressed Arabidopsis thaliana. However, under salt stress, KCR heterologously overexpressed Arabidopsis thaliana plants showed obvious salt stress tolerance phenotypes. The seed germination rate and the main root length were significantly higher than those of the wild type, while the leaf wilting rate of Arabidopsis thaliana adult plants was significantly lower than that of the wild type ( Figure 9 A-C in).

[0118] From Figure 10 It can be seen that after salt stress treatment, compared with the wild type, KCR heterologous overexpression of Arabidopsis thaliana increased the accumulation of leaf wax crystals ( Figure 10 A, the arrow indicates the wax crystals). The content of VLCFAs increased significantly, and the content of VLCFA components, especially C24 and C26, increased significantly ( Figure 10 B), and KCR the accumulation of epidermal wax in the leaves of heterologous overexpression Arabidopsis thaliana increased significantly, and the content of each wax component increased.

[0119] The GC-MS detection results also showed that after salt treatment, KCR the accumulation of epidermal wax in the leaves of heterologous overexpression Arabidopsis thaliana increased significantly, and the content of each wax component increased. Among them, the increase in alkanes was obvious ( Figure 10 C).

[0120] The above results indicate that KCR regulates the synthesis of VLCFAs and epidermal wax and participates in the process of plant resistance to salt stress.

[0121] From Figure 11 it can be seen that when detecting KCR the water status of heterologous overexpression Arabidopsis thaliana, under normal conditions, the relative water content of the wild type and the overexpression Arabidopsis thaliana lines did not differ significantly ( Figure 11 A, the first from the left in each group is the relative water content of the wild type Arabidopsis thaliana without salt treatment, and the second, third, and fourth are the relative water contents of the heterologous overexpression OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis thaliana, the fifth is the relative water content of the wild type Arabidopsis thaliana with salt treatment, and the sixth, seventh, and eighth are the relative water contents of the heterologous overexpression OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis thaliana). After salt stress, the leaf water loss rate of the three overexpression lines was lower than that of the wild type Arabidopsis thaliana. At 5 h of salt stress, the leaf water loss rate of the wild type Arabidopsis thaliana reached 50%, and the leaf water loss rate of the overexpression lines was between 30% and 40% ( Figure 11 B); the relative water content of the overexpression lines was significantly higher than that of the wild type ( Figure 11 A).

[0122] From Figure 12 it can be seen that salt stress increased the relative permeability of the cell membrane in Arabidopsis thaliana leaves and promoted the content of MDA in Arabidopsis thaliana leaves. However, the relative permeability of the cell membrane and MDA in the overexpression lines were significantly lower than those in the WT ( Figure 12 A and Figure 12B. For each group, the first one from the left is the relative membrane permeability and MDA content of wild-type Arabidopsis thaliana without salt treatment. The second, third, and fourth ones are the relative membrane permeability and MDA content of heterologous overexpression OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis thaliana. The fifth one is the relative membrane permeability and MDA content of wild-type Arabidopsis thaliana under salt treatment. The sixth, seventh, and eighth ones are the relative membrane permeability and MDA content of heterologous overexpression OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis thaliana). In overexpression lines, the activities of SOD, POD, CAT and the content of superoxide anion are significantly higher than those of wild-type ( Figure 12 C-F. For each group, the first one from the left is the content of superoxide anion, SOD activity, POD activity and CAT activity of wild-type Arabidopsis thaliana without salt treatment. The second, third, and fourth ones are the content of superoxide anion, SOD activity, POD activity and CAT activity of heterologous overexpression OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis thaliana. The fifth one is the content of superoxide anion, SOD activity, POD activity and CAT activity of wild-type Arabidopsis thaliana under salt treatment. The sixth, seventh, and eighth ones are the content of superoxide anion, SOD activity, POD activity and CAT activity of heterologous overexpression OEKCR-3 , OEKCR-7 and OEKCR-9 Arabidopsis thaliana).

[0123] The above results show that under salt stress, KCR can reduce water loss and maintain plant water content, and by increasing the antioxidant enzyme activity in Arabidopsis thaliana leaves, reduce the accumulation of reactive oxygen species and alleviate the membrane oxidative damage caused by salt stress.

[0124] It should be noted that the specific implementation manners are only relatively representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and there can be many variations. Those of ordinary skill in the art who obtain without any doubt according to what is clearly disclosed in the present invention or according to the written description of the document shall be considered as the scope to be protected by this patent.

Claims

1. Use of the β-ketoacyl-CoA reductase KCR gene in enhancing plant salt tolerance, characterized in that, The nucleotide sequence of the β-ketoacyl-CoA reductase KCR gene is shown in SEQ ID NO.1; the plant is grape or Arabidopsis thaliana.

2. The application according to claim 1, wherein The application is at least one of the following (1) to (3): (1) Transient overexpression of the KCR gene in plants significantly increases the accumulation of phenols, wax esters, and alkanes in leaves; (2) Overexpression of the KCR gene in grape callus. Under salt stress, the overexpressed grape callus has increased SOD, POD, and CAT activities compared with the wild-type grape callus; the content of MDA, the accumulation of H2O2 and superoxide anion are reduced; (3) Overexpression of the KCR gene in grape callus. Under salt stress, the expression levels of salt-responsive genes CAX, HATP, HKT2, HPP, SOS1, and SORK are significantly increased.

3. Application of β-ketoacyl-CoA reductase KCR gene in genetic improvement of plant salt tolerance, characterized in that, The nucleotide sequence of the β-ketoacyl-CoA reductase KCR gene is shown in SEQ ID NO.1; the plant is grape or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The application is at least one of the following (a) to (b): (a) Overexpression of the KCR gene in callus, and the callus is induced to form seedlings; (b) Overexpression of the KCR gene in plants, and the offspring are propagated and screened.

5. A method for improving the salt tolerance of plants, characterized in that, The β-ketoacyl-CoA reductase KCR gene is constructed into an expression vector to form a recombinant expression vector, and then the recombinant expression vector is transformed into a strain to obtain a recombinant strain carrying the KCR gene. Then the recombinant strain is used to infect the plant, so that the plant carries the KCR gene. Finally, through the expression of the KCR gene, the accumulation of epidermal wax and the biosynthesis of VLCFAs are regulated, thereby regulating the salt tolerance of the plant; the nucleotide sequence of the β-ketoacyl-CoA reductase KCR gene is shown in SEQ ID NO.1; the plant is grape or Arabidopsis thaliana.

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